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M P Yaffe

Publications and source records attributed to M P Yaffe.

18 recordsLinked to original sources
PubMed1992

Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein.

The mdml mutation causes temperature-sensitive growth and defective transfer of nuclei and mitochondria into developing buds of yeast cells at the nonpermissive temperature. The MDM1 gene was cloned by complementation, and its sequence revealed an open reading frame encoding a potential protein product of 51.5 kD. This protein displays amino acid sequence similarities to hamster vimentin and mouse epidermal keratin. Gene disruption demonstrated that MDM1 is essential for mitotic growth. Antibodies against the MDM1 protein recognized a 51-kD polypeptide that was localized by indirect immunofluorescence to a novel pattern of spots and punctate arrays distributed throughout the yeast cell cytoplasm. These structures disappeared after shifting mdm1 mutant cells to the nonpermissive temperature, although the cellular level of MDM1 protein was unchanged. Affinity-purified antibodies against MDM1 also specifically recognized intermediate filaments by indirect immunofluorescence of animal cells. These results suggest that novel cytoplasmic structures containing the MDM1 protein mediate organelle inheritance in yeast.

Amino Acid Sequence↗
PubMed1992

MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import.

The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae. The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene. The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1. Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures. Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures. The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C. These results indicate a role for a DnaJ cognate protein in mitochondrial protein import.

Amino Acid Sequence↗
PubMed1991

Uncoupling thermotolerance from the induction of heat shock proteins.

Exposure of cells to elevated temperatures causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear. hsp induction in the yeast Saccharomyces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsf1-m3, in heat-shock transcription factor that prevents the factor's activation. We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance.

Base Sequence↗
PubMed1991

A role for unsaturated fatty acids in mitochondrial movement and inheritance.

Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.

Cloning, Molecular↗
PubMed1991

A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle.

Yeast cells containing the recessive mas3 mutation display temperature-sensitive defects in both mitochondrial protein import and the cell division cycle. The import defect is characterized by two pools of mitochondrial precursors and a dramatically slower rate of posttranslational import. The effect of mas3 on cell cycle progression occurs within one cell cycle at the nonpermissive temperature and retards progression through the G2 stage. The mas3 mutation maps to the gene encoding yeast heat-shock transcription factor (HSF), and expression of wild-type HSF complements the temperature-sensitive defects. The mas3 lesion has no apparent effect on protein secretion. In mas3 cells, induction of a major heat-shock gene, SSA1, is defective at 37 degrees C. The properties of the mas3 mutant cells indicate that HSF mediates the response to stress of two basic cellular processes: mitochondrial protein import and cell cycle progression.

Base Sequence↗
PubMed1990

A yeast protein, homologous to the proteolipid of the chromaffin granule proton-ATPase, is important for cell growth.

We have characterized a gene, PPA1, adjacent to the yeast MAS2 gene. DNA sequence analysis of PPA1 predicts a hydrophobic protein of 23 kDa. This protein is homologous to the proteolipid of the bovine chromaffin granule proton ATPase and to the proteolipid of the yeast vacuolar proton ATPase. Gene disruption experiments indicate that the PPA1 protein is essential for viability in three unrelated yeast strains and important for optimal growth in a fourth strain.

Adenosine Triphosphatases↗
PubMed1990

Embedded or not? Hydrophobic sequences and membranes.

The same translocation machinery appears to be responsible for both the translocation of soluble proteins across membranes and the insertion of integral membrane proteins into the bilayer. A single mechanism is proposed to accommodate these two functions. This model is also extended to explain the paradoxical translocation of mitochondrial and chloroplastic membrane proteins across one or more membranes before they are finally inserted into their target membranes.

Amino Acid Sequence↗
PubMed1990

Temperature-sensitive yeast mutants defective in mitochondrial inheritance.

The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

Actins↗
PubMed1989

The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function.

As part of an analysis of the function and assembly of the mitochondrial outer membrane, we have cloned and characterized the yeast gene encoding a 45-kDa polypeptide (OM45) which is a major constituent of this membrane. The nuclear gene was isolated by immunological screening of plaques of recombinant phage lambda gt11 containing fragments of yeast genomic DNA using an antibody against OM45. Determination of the nucleotide sequence of the DNA fragment isolated by this approach revealed a single open reading frame of 1179 base pairs which encodes a protein having a predicted molecular mass of 44.6-kDa. Disruption of the OM45 gene in haploid yeast cells eliminated the expression of OM45. The mutant strain showed no apparent defect in cell viability, growth, mitochondrial function, or mitochondrial protein import.

Amino Acid Sequence↗
PubMed1988

Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes.

We have purified the metalloprotease which is localized in the soluble matrix space of Saccharomyces cerevisiae mitochondria and cleaves the amino-terminal matrix-targeting sequences from imported mitochondrial precursor proteins. The enzyme consists of two loosely associated non-identical subunits of mol. wt 48,000 and 51,000, respectively. Attempts to separate the two subunits from each other caused loss of activity. The smaller subunit had been identified as the product of the nuclear MAS1 gene (Witte et al., 1988). The larger subunit is now identified as the product of the nuclear MAS2 gene.

Alcohol Dehydrogenase↗
PubMed1988

Import of proteins into yeast mitochondria: the nuclear MAS2 gene encodes a component of the processing protease that is homologous to the MAS1-encoded subunit.

The mas2 mutant of Saccharomyces cerevisiae is temperature sensitive for import of proteins into mitochondria. To identify the lesion in this mutant, we have cloned and sequenced the wild-type MAS2 gene and determined the intracellular location of its protein product. MAS2 encodes an essential 53-kd protein that is located in the mitochondrial matrix and is homologous to the MAS1 protein, a previously identified subunit of the protease that cleaves presequences from mitochondrial precursor proteins. The activity of this enzyme is temperature sensitive in mas2 cells. Together with the results of the accompanying study these results show that MAS2 and MAS1 encode the two subunits of the processing protease.

Amino Acid Sequence↗
PubMed1988

MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease.

We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.

Amino Acid Sequence↗
PubMed1987

On the translocation of proteins across membranes.

Many proteins of intracellular organelles are first synthesized in the cytoplasm and are then specifically transferred across the membranes of the organelles. On the assumption that these transfers all occur by the same basic mechanism, we enumerate the rather stringent requirements that the mechanism must satisfy. A unitary molecular mechanism is then proposed that meets these requirements.

Amino Acid Sequence↗
PubMed1987

On the transfer of integral proteins into membranes.

We have earlier proposed a molecular mechanism for the translocation of hydrophilic proteins across membranes that accounts for the experimental facts and meets the restrictions that we stipulate for such a mechanism. In particular, the restrictions are that translocation occurs by successive segments of the polypeptide chain and that the ionic groups of the polypeptide remain in contact with water throughout the translocation process. The evidence indicates that the transfer of integral proteins into membranes very likely uses the same molecular machinery as does the translocation of hydrophilic proteins across membranes. Here we show how the mechanism we have proposed for translocation can also be utilized in the intercalation of known types of integral proteins, accounting for their specific topologies in the membrane.

Biological Transport↗
PubMed1985

A yeast mutant temperature-sensitive for mitochondrial assembly is deficient in a mitochondrial protease activity that cleaves imported precursor polypeptides.

We have previously described two yeast mutants which, at elevated temperature, stop growing and accumulate precursors to several imported mitochondrial proteins. We now show that one of these mutants (mas 1) is deficient in a matrix-located protease activity which cleaves the pre-sequences from mitochondrial precursor proteins. Isolated mas 1 mitochondria catalyze oxidative phosphorylation, exhibit respiratory control and import mitochondrial precursor polypeptides, but are defective in removing transient pre-sequences from imported precursors. The phenotype of the mas 1 mutant suggests that the matrix-located processing protease is essential for growth and for mitochondrial assembly.

Adenosine Diphosphate↗
PubMed1984

Two nuclear mutations that block mitochondrial protein import in yeast.

We isolated two yeast mutants that are temperature-sensitive for import of mitochondrial proteins. Each strain contains a single mutation that results in arrest of growth and accumulation of precursor to the beta subunit of the mitochondrial F1-ATPase after incubation at 37 degrees C. These lesions (mas1 and mas2) are nonallelic and recessive. Cells harboring either mutation stop growing only after 2-3 generations at 37 degrees C. Import of the F1 beta subunit at 37 degrees C is more than 250 times slower in mas1 and 15 times slower in mas2 than in wild-type cells. At 23 degrees C, import occurs with similar rates in mutant and wild-type cells. The two mutations also reduce the rate of import of other proteins; however, import of different precursors is affected to different degrees in the two strains. The temperature-sensitive step in import in both mas1 and mas2 occurs before arrival of precursors in the mitochondrial matrix.

Biological Transport↗
PubMed1979

Biosynthesis of phospholipids in Bacillus megaterium.

Information on the biosynthesis of phospholipids in bacteria has been derived principally from the study of Escherichia coli and other gram-negative organisms. We have now carried out a detailed study of the pathways of phospholipid biosynthesis in the gram-positive organism Bacillus megarterium KM in relation to investigations on the biogenesis of lipid asymmetry in membranes. Radioactive precursors such as 32Pi and [3H]palmitate initially label phosphatidylethanolamine much more than phosphatidylglycerol. This raised the possibility that phosphatidylglycerol may be the precursor of phosphatidylethanolamine in a pathway different from that in E. coli. Phosphatidylglycerol is known to be highly reactive metabolically, since it functions as a donor of phosphatidyl residues in the synthesis of cardiolipin and as a donor of glycerophosphate residues in the synthesis of teichoic acids and of membrane-derived oligosaccharides. The large pool of phosphatidylglycerol would dilute the radioactive isotope, slowing the initial rate of incorporation of label into phosphatidylethanolamine. However, assays of cell-free extracts revealed no evidence for such a novel pathway. Instead, phosphatidylserine synthase (cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase) and phosphatidylserine decarboxylase were detected, although at low levels. These results suggest that the pathway in B. megaterium is the same as that in E. coli in which phosphatidylserine, derived from cytidine 5'-diphosphate-diglyceride, is the precursor of phosphatidylethanolamine. The lag in the appearance of label in phosphatidylethanolamine appears to be the effect of a considerable pool of phosphatidylserine (ca. 5 to 10% of the total phospholipid) in certain strains of B. megaterium. The lag in labeling can be correlated with the size of the pool of phosphatidylserine. Pulse-chase experiments in vivo support the conclusion that in B. megaterium phosphatidylserine is not derived from phosphatidylglycerol. Rates of turnover of the membrane phospholipids of B. megaterium have also been studied.

Bacillus megaterium↗